FLT3 WT overexpression also abolishes hematopoietic differentiation from MLL-AF4-hESCs. (A) Phase-contrast morphology (top) and fluorescence microscopy (bottom) of colonies from MLL-AF4/FLT3-WT-hESCs. (B) Quantitative RT-PCR confirming efficient transduction and stable expression of FLT3-WT. (C) Phosphosignaling analysis of transduced hESCs, showing increased AKT, ERK, and STAT5 phosphorylation in cells transduced with FLT3-WT. FLT3-WT slightly impairs specification into hemogenic precursors of MLL-AF4–expressing hESCs (D), whereas it completely abolishes differentiation of MLL-AF4–expressing hESCs into primitive blood cells (E) and total blood cells (F). (G) CFU readout from day 15 hEBs confirms a significant decrease in hematopoietic potential in hESCs coexpressing MLL-AF4 and FLT3-WT. Data are presented as mean ± SEM for 5 independent experiments.